The severe postnatal loss of -cells and diabetes in insulin receptor substrate 2 deficient mice (Irs2-/-) has confirmed the necessity of maintaining -cell insulin signaling for adaptive expansion and survival in the context of insulin resistance. Previous work in several cell types and some in -cells has established the anti-proliferative and pro- apoptotic effects of FoxO and Gsk3 proteins that are negatively regulated by insulin signaling. Haplo-insufficiency of FoxO1 has been shown to partially restore -cell mass in Irs2-/- mice. Our preliminary studies in Irs2-/- mice now show that haplo-insufficiency of Gsk3 markedly increases -cell proliferation and survival, maintaining -cell mass and also preventing diabetes. However, there is still little information about -cell specific effects of Gsk3, or about functional consequences of a deficiency of either Gsk3 or FoxO proteins in -cells. To address this we have generated three -cell specific mouse models with: 1) over-expression of a constitutively active Gsk3 (RIP-Gsk3CA), 2) conditional deletion of the Gsk3 gene (-Gsk3KO), and 3) expression of a loss-of-function mutant of FoxO1 (RIP-FoxO?256). In this application we describe experiments that utilize these models and in vitro cell systems to test the hypothesis that diminished insulin signaling (i.e. resistance) in the -cell leads to increased activity of FoxO1 and GSK3, two anti-proliferative and pro-apoptotic proteins that work in concert to promote loss of -cell mass. The first Specific Aim is to determine the role of Gsk3 in -cell expansion and survival by characterizing -cell specific Gsk3 deficient (-Gsk3KO) mice, in -Gsk3KO mice crossed with Irs2-/- insulin resistant mice, and to define Gsk3 downstream molecular mechanisms with combined in vitro and in vivo models of altered Gsk3 activity. Another aim is to examine the role of FoxO proteins in -cell expansion and survival by characterizing -cell specific loss-of-function FoxO1 mice (RIP-FoxO?256), by examining the Irs2-/- mouse crossed with the RIP-FoxO?256 mouse, and to assess FoxO downstream molecular mechanisms with an in vitro model of -cell insulin resistance. In the last aim the effects of individual and combined deficiencies of -cell Gsk3 and FoxO proteins on the adaptive expansion of -cell mass to diet induced obesity will be assessed. Several questions will be addressed in these models: 1) what are the roles of Gsk3 and FoxO proteins in the response of -cells to metabolic stress? 2) Will suppression of these proteins prevent some forms of diabetes and are the effects additive? 3) What are the downstream effectors and are some of them shared by Gsk3 and FoxO proteins? The anticipated results of these studies may be beneficial in averting diabetes onset in obese insulin resistant individuals.